This invention relates to a method for producing human intervertebral disc cells. More particularity, this invention relates to methods for growing intervertebral cells in monolayers cultures followed by three-dimensional growth of cells.
In spite of the large health care costs associated with degenerative disc disease, the cell biology of the human intervertebral disc cell has been neglected compared to the knowledge available on chondrocytes or bone cell populations. Recent studies have presented data on the formation and turnover of matrix in situ in the human disc. Antoniou, J., Steffen, T., Nelson, F., Winterbottom, N., Hollander, A. P., Poole, R. A., Aebi, M., Alini, M., “The human lumbar intervertebral disc-Evidence for changes in the biosynthesis and denaturation of the extra cellular matrix with growth, maturation, ageing, and degeneration,” J. Clin. Invest. 98, 996–1003 (1996). Growth of the young disc showed active matrix formation and denaturation of Type II collagen; aging and maturation were associated with decreased matrix synthesis and reduced denaturation of Type II collagen. Degenerative stages showed decreased aggrecan and Type II procollagen formation and increased Type II denaturation and Type I collagen synthesis. Successful isolation and in vitro growth of disc cells under experimental conditions can be a valuable tool for clarification of the cellular mechanisms involved in these observed matrix changes.
Studies of human disc cells cultured in alginate beads have provided evidence that more than one distinctive cell population resides in the disc. Chelberg, M. K., Banks, G. M., Geiger, D. F., Oegema, T. R., “Identification of heterogeneous cell populations in normal human intervertebral disc,” J Anat, 186, 43–53 (1995). Others have used the alginate bead technique to study canine disc cells in culture. Maldonado, B. A., Oegema, T. R., “Initial characterization of the metabolism of intervertebral disc cells encapsulated in microspheres,” J Orthopaedic Res, 10, 677–690 (1992). Cells from the rat disc have been grown in monolayer culture. Ichimura, K., Tsuji, H., Matsui, H., Makiyama, N., Cell culture of the intervertebral disc of rats: Factors influencing culture, proteoglycan, collagen, and deoxyribonucleic acid synthesis,” J Spinal Disorders, 4, 428–436 (1991).
Three-dimensional cell culture is a preferred culture method for chondrocytes, a cell type similar to at least some members of the disc cell population, and is known to de-differentiate in monolayer culture and reexpress a characteristic Type II collagen extracellular matrix production when placed in agarose culture. Benya, P. D., Shaffer, J. D., “Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultures in agarose gels,” Cell, 30, 215–224 (1982).
It is an object of the present invention to produce human intervertebral cells in monolayer explant cultures.
Another object of the present invention is to produce human intervertebral cells in a three-dimensional cultures.